Unprecedented forms of vanadium observed within the blood cells of Phallusia nigra using K-edge X-ray absorption spectroscopy.

Fits to the vanadium K-edge X-ray absorption spectra (XAS) of five whole blood cell samples from the tunicate Phallusia nigra revealed unprecedented forms of intracellular vanadium. Endogenous vanadium was divided between the V(III) ion (74.2+/-5.1% of total V) and the vanadyl ion [V(IV)=O](2+) (25.2+/-5.4% of total V). The V(III) fraction included both [V(H(2)O)(6)](3+) (36.7+/-5.5%) modeled as VCl(3) in 1 M HCl, and three previously unprecedented chelated V(III) forms (37.5+/-4.6%). Two of these could be represented by the model ligand environments V(acetylacetonate)(3) (17.9+/-3.2%) and K(3)V(catecholate)(3) (13.1+/-4.7%), implying DOPA-like complexation. The third chelated form was represented by the 7-coordinate N(2)O(5) complex Na[V(edta)(H(2)O)] (8.0+/-1.8%). This coordination array, suggestive of a novel mononuclear V(III) protein site, contributed only to fits to samples 1, 2, 3 and 5, which were prepared in the presence of DTT. Endogenous V(IV) (25.2+/-5.4%) was principally modeled as VOCl(2) in 1 M HCl. EPR spectra (averages: A(parallel)=(1.842+/-0.006)x10(-2) cm(-1); A( perpendicular)=(0.718+/-0.007)x10(-2) cm(-1); g(parallel)=1.936+/-0.002; g( perpendicular)=1.990+/-0.001) confirmed the predominance of the aquated vanadyl ion. Blood cell sample five uniquely required the XAS spectrum of VOSO(4) in 0.1 M H(2)SO(4) solution (13.0%) and of [OV(V)(pivalate)(3)] (3.1%) to successfully fit the XAS pre-edge energy region. This endogenous V(V) signal is also unprecedented. These results are compared with those of analogous fits to the blood cells of Ascidia ceratodes and may support assignment of P. nigra to a different genus.

Determination of ligand binding constants for the iron-molybdenum cofactor of nitrogenase: monomers, multimers, and cooperative behavior.

Equilibrium titrations in N-methylformamide (NMF) of G-25 gel filtered (ox)-state FeMo cofactor [FeMoco(ox)] from Azotobacter vinelandii nitrogenase were carried out using sodium ethanethiolate and followed using UV/Vis absorption spectroscopy. For Fe-Moco(ox), a non-linear least squares (NLLSQ) fit to the data indicated a strong equilibrium thiolate-binding step with Keq = 1.3+/-0.2x10(6) M(-1). With 245 molar excess imidazole, cooperative binding of three ethanethiolates was observed. The best NLLSQ fit gave Keq=2.0+/-0.1x10(5) M(-2) and a Hill coefficient n=2.0+/-0.3. A Scatchard plot of these data was concave upward, indicating positive cooperativity. The fit to previously published data involving benzenethiol titration of the one-electron reduced (semi-reduced) cofactor, FeMoco(sr), as followed by EPR required a model that included both a sub-stoichiometric ratio of thiol to FeMoco(sr) and about five cooperative ligand binding sites. These constraints were met by modeling FeMoco(sr) as an aggregate, with fewer thiol binding sites than FeMoco(sr) units. The best fit model was that of FeMoco(sr) as a dodecamer with five cooperative benzenethiol binding sites, yielding a thiol binding constant of 3.32+/-0.09x10(4) M(-4.8) and a Hill coefficient n=4.8+/-0.6. The results of all the other published ligand titrations of FeMoco(sr) were similarly analyzed successfully in terms of equilibrium models that include both cooperative ligand binding and dimer-level aggregation. A possible structural model for FeMoco aggregation in NMF solution is proposed.

Synthesis and spectroscopic studies of non-heme diiron(III) species with a terminal hydroperoxide ligand: models for hemerythrin.

Two compounds, [Fe2(mu-OH)(mu-Ph4DBA)(TMEDA)2(OTf)] (4) and [Fe2(mu-OH)(mu-Ph4DBA)(DPE)2(OTf)] (7), where Ph4DBA(2-) is the dinucleating bis(carboxylate) ligand dibenzofuran-4,6-bis(diphenylacetate), have been prepared as synthetic models for the dioxygen-binding non-heme diiron protein hemerythrin (Hr). X-ray crystallography reveals that, in the solid state, these compounds contain the asymmetric coordination environment found at the diiron center in the reduced form of the protein, deoxyHr. Mössbauer spectra of the models (4, delta = 1.21(2), DeltaE(Q) = 2.87(2) mm s(-1); 7, delta(av) = 1.23(1), DeltaE(Qav) = 2.79(1) mm s(-1)) and deoxyHr (delta = 1.19, DeltaE(Q) = 2.81 mm s(-1)) are also in good agreement. Oxygenation of the diiron(II) complexes dissolved in CH2Cl2 containing 3 equiv of N-MeIm (4) or neat EtCN (7) at -78 degrees C affords a red-orange solution with optical bands at 336 nm (7300 M(-1) cm(-1)) and 470 nm (2600 M(-1) cm(-1)) for 4 and at 334 nm (6400 M(-1) cm(-1)) and 484 nm (2350 M(-1) cm(-1)) for 7. These spectra are remarkably similar to that of oxyHr, 330 nm (6800 M(-1) cm(-1)) and 500 nm (2200 M(-1) cm(-1)). The electron paramagnetic resonance (EPR) spectrum of the cryoreduced, mixed-valence dioxygen adduct of 7 displays properties consistent with a (mu-oxo)diiron(II,III) core. An investigation of 7 and its dioxygen-bound adduct by extended X-ray absorption fine structure (EXAFS) spectroscopy indicates that the oxidized species contains a (mu-oxo)diiron(III) core with iron-ligand distances in agreement with those expected for oxide, carboxylate, and amine/hydroperoxide donor atoms. The analogous cobalt complex [Co2(mu-OH)(mu-Ph4DBA)(TMEDA)2(OTf)] (6) was synthesized and structurally characterized, but it was unreactive toward dioxygen.

S K-edge X-ray absorption studies of tetranuclear iron-sulfur clusters: mu-sulfide bonding and its contribution to electron delocalization.

X-ray absorption spectroscopy (XAS) at the sulfur ( approximately 2470 eV) and chlorine ( approximately 2822 eV) K-edges has been applied to a series of 4Fe-4S model complexes. These are compared to 2Fe-2S model complexes to obtain insight into the localized ground state in the mixed-valence dimer versus the delocalized ground state in the mixed-valence tetramer. The preedges of hypothetical delocalized mixed-valence dimers [Fe(2)S(2)](+) are estimated using trends from experimental data and density functional calculations, for comparison to the delocalized mixed-valence tetramer [Fe(4)S(4)](2+). The differences between these two mixed-valence sites are due to the change of the sulfide-bridging mode from micro(2) to micro(3). The terminal chloride and thiolate ligands are used as spectator ligands for the electron density of the iron center. From the intensity of the preedge, the covalency of the terminal ligands is found to increase in the tetramer as compared to the dimer. This is associated with a higher effective nuclear charge on the iron in the tetramer (derived from the energies of the preedge). The micro(3)-bridging sulfide in the tetramer has a reduced covalency per bond (39%) as compared to the micro(2)-bridging sulfide in the dimer (51%). A simple perturbation model is used to derive a quadratic dependence of the superexchange coupling constant J on the covalency of the metal ions with the bridging ligands. This relationship is used to estimate the superexchange contribution in the tetramer (J = -156 cm(-)(1)) as compared to the mixed-valence dimer (J = -360 cm(-)(1)). These results, combined with estimates for the double exchange and the vibronic coupling contributions of the dimer sub-site of the tetramer, lead to a delocalized S(t) = (9)/(2) spin ground state for the mixed-valence dimer in the tetramer. Thus, the decrease in the covalency, hence the superexchange pathway associated with changing the bridging mode of the sulfides from micro(2) to micro(3) on going from the dimer to the tetramer, significantly contributes to the delocalization of the excess electron over the dimer sub-site in the tetramer.

Sulfur K-edge X-ray absorption spectroscopy of 2Fe-2S ferredoxin: covalency of the oxidized and reduced 2Fe forms and comparison to model complexes.

Ligand K-edge X-ray absorption spectroscopy (XAS) provides a direct experimental probe of ligand-metal bonding. In previous studies, this method has been applied to mononuclear Fe-S and binuclear 2Fe-2S model compounds as well as to rubredoxins and the Rieske protein. These studies are now extended to the oxidized and reduced forms of ferredoxin I from spinach. Because of its high instability, the mixed-valence state was generated electrochemically in the protein matrix, and ligand K-edge absorption spectra were recorded using an XAS spectroelectrochemical cell. The experimental setup is described. The XAS edge data are analyzed to independently determine the covalencies of the iron-sulfide and -thiolate bonds. The results are compared with those obtained previously for the Rieske protein and for 2Fe-2S model compounds. It is found that the sulfide covalency is significantly lower in oxidized FdI compared to that of the oxidized model complex. This decrease is interpreted in terms of H bonding present in the protein, and its contribution to the reduction potential E degrees is estimated. Further, a significant increase in covalency for the Fe(III)-sulfide bond and a decrease of the Fe(II)-sulfide bond are observed in the reduced Fe(III)Fe(II) mixed-valence species compared to those of the Fe(III)Fe(III) homovalent site. This demonstrates that, upon reduction, the sulfide interactions with the ferrous site decrease, allowing greater charge donation to the remaining ferric center. That is the dominant change in electronic structure of the Fe(2)S(2)RS(4) center upon reduction and can contribute to the redox properties of this active site.

A quantitative description of the ground-state wave function of Cu(A) by X-ray absorption spectroscopy: comparison to plastocyanin and relevance to electron transfer.

To evaluate the importance of the electronic structure of Cu(A) to its electron-transfer (ET) function, a quantitative description of the ground-state wave function of the mixed-valence (MV) binuclear Cu(A) center engineered into Pseudomonas aeruginosa azurin has been developed, using a combination of S K-edge and Cu L-edge X-ray absorption spectroscopies (XAS). Parallel descriptions have been developed for a binuclear thiolate-bridged MV reference model complex ([(L(i)(PrdacoS)Cu)(2)](+)) and a homovalent (II,II) analogue ([L(i)(Pr2tacnS)Cu)(2)](2+), where L(i)(PrdacoS) and L(i)(Pr2tacnS) are macrocyclic ligands with attached thiolates that bridge the Cu ions. Previous studies have qualitatively defined the ground-state wave function of Cu(A) in terms of ligand field effects on the orbital orientation and the presence of a metal--metal bond. The studies presented here provide further evidence for a direct Cu--Cu interaction and, importantly, experimentally quantify the covalency of the ground-state wave function. The experimental results are further supported by DFT calculations. The nature of the ground-state wave function of Cu(A) is compared to that of the well-defined blue copper site in plastocyanin, and the importance of this wave function to the lower reorganization energy and ET function of Cu(A) is discussed. This wave function incorporates anisotropic covalency into the intra- and intermolecular ET pathways in cytochrome c oxidase. Thus, the high covalency of the Cys--Cu bond allows a path through this ligand to become competitive with a shorter His path in the intramolecular ET from Cu(A) to heme a and is particularly important for activating the intermolecular ET path from heme c to Cu(A).

An approach to three-dimensional structures of biomolecules by using single-molecule diffraction images.

We describe an approach to the high-resolution three-dimensional structural determination of macromolecules that utilizes ultrashort, intense x-ray pulses to record diffraction data in combination with direct phase retrieval by the oversampling technique. It is shown that a simulated molecular diffraction pattern at 2.5-A resolution accumulated from multiple copies of single rubisco biomolecules, each generated by a femtosecond-level x-ray free electron laser pulse, can be successfully phased and transformed into an accurate electron density map comparable to that obtained by more conventional methods. The phase problem is solved by using an iterative algorithm with a random phase set as an initial input. The convergence speed of the algorithm is reasonably fast, typically around a few hundred iterations. This approach and phasing method do not require any ab initio information about the molecule, do not require an extended ordered lattice array, and can tolerate high noise and some missing intensity data at the center of the diffraction pattern. With the prospects of the x-ray free electron lasers, this approach could provide a major new opportunity for the high-resolution three-dimensional structure determination of single biomolecules.

Ligand K-edge X-ray absorption spectroscopy: a direct probe of ligand-metal covalency.

Ligand K-edge X-ray absorption spectroscopy (XAS) is a new experimental probe of the covalency of a metal-ligand bond. The intensity of the ligand pre-edge feature is proportional to the mixing of ligand orbitals into the metal d orbitals. The methodology to determine covalencies in one-electron (hole) and many-electron systems is described and demonstrated for a series of metal tetrachlorides [MCl(4)](n)(-), metal tetrathiolates [M(SR)(4)](n)(-), and dimeric iron-sulfur (Fe-S) clusters [Fe(2)S(2)(SR)(4)](2-). It is then applied to blue Cu proteins, the Cu(A) site, hydrogen bonding in Fe-S clusters, and the delocalization behavior in [2Fe-2S] vs [4Fe-4S] clusters. The covalencies determined in these studies provide important electronic structure insight into function.

Defining chemical species in complex environments using K-edge X-ray absorption spectroscopy: vanadium in intact blood cells and Henze solution from the tunicate Ascidia ceratodes.

A K-edge X-ray absorption spectrum (XAS) fitting approach has been developed to speciate elements of interest in complex materials and used here to model the storage of biological vanadium within whole blood cells from the tunicate Ascidia ceratodes. The response of the K-edge XAS of solution-phase V(III) to increasing c(sulfate) at constant pH 1.8 produced specific and systematic effects in the preedge transition at 5468.8 eV (preedge transitions: 1s-->4A2 at 5464.9 +/- 0.1 eV, 1s-->4T2 at 5466.9 +/- 0.1 eV, and 1s-->4T1 at 5468.8 +/- 0.1 eV for 11 different V(III)/sulfate solutions). In contrast, variations in acidity (as pH) at constant c(sulfate) systematically modified the V(III) preedge XAS at 5466.9 eV. The energy position of the K-edge absorption maximum also serially shifted -0.32 eV/pH unit, from 5483.7 eV (pH 3.0) to 5484.7 eV (pH 0.3). Fits to the V-K XAS of two samples of A. ceratodes whole blood cells representing dozens of animals implied storage of V(III) ions in four predominant solution regimes: approximately 10% high sulfate/pH 0 acid; approximately 40% high sulfate/pH 1.8 acid; approximately 40% moderate sulfate/pH 1.8 acid; approximately 10% moderate sulfate/pH 3 acid. For lysed blood cells, the best fit represented 63% of the V(III) in a pH 1.6 sulfate-free environment and a further 16% in acidic sulfate solution. Nearly 18% of lysed cells vanadium(III) appeared in a tris(catecholate)-like environment. A detailed speciation of biological vanadium complex ions was calculated from these fits by application of the known equilibrium constants governing V(III) and sulfate in acidic aqueous solution. The utility of blood cell V(III) to ascidians is discussed. Fits to K-edge XAS spectra using the XAS spectra of appropriate models are suggested to be generally applicable to elucidating the state of metal ions in a wide variety of complex environments.

Metallocyclopeptide complexes with MII(S.Cys)4 chromophores.

The tetracysteinyl peptide cyclo[Lys1,12](Gln-Cys-Gly-Val-Cys-Gly-Lys-Cys-Ile-Ala-Cys-Lys) ([symbol: see text] L(Cys.SH)4) was synthesized by solid-phase methods using an Fmoc/t-Bu/allyl strategy on a PAL-PEG-PS support. The formation of the 1:1 complexes with M = Fe2+, Co2+, and Ni2+ was observed by spectrophotometric monitoring of reactions in aqueous solution at pH 7.5. Size exclusion chromatography indicated that the peptide is a monomer and the complexes are dimers [M2([symbol: see text]L(Cys.S)4)2] in aqueous buffer at pH 7.5. Cobalt and nickel K-edge X-ray absorption data and EXAFS analysis of [Co2([symbol: see text] L(Cys.S)4)2] and [Ni2([symbol: see text] L(Cys.S)4)2] as lyophilized solids are reported. Derived bond distances are Co-S = 2.30 A and Ni-S = 2.21 A. From the collective results provided by absorption spectra, K-edges, EXAFS, and bond length comparisons with known structures, it is shown that [Fe2([symbol: see text] L(Cys.S)4)2] and [Co2([symbol: see text] L(Cys.S)4)2] possess distorted tetrahedral structures and [Ni2([symbol: see text] L(Cys.S)4)2] has distorted square planar stereochemistry. The Co(II) chromophore is particularly distinctive of the assigned structure, displaying three components of the parent tetrahedral ligand field transition 4A2-->4T1(P) (610, 685, 740 nm). The observed structures conform to the intrinsic stereochemical preferences of the metal ions. Structures for the binuclear complexes are suggested. These are the first characterized metal complexes of a cysteinyl cyclopeptide and among the few well-documented complexes of synthetic cyclopeptides. This study is a desirable first step in the design of cyclic peptides for the binding of mononuclear and polynuclear metal centers.

X-ray spectroscopy of enzyme active site analogues and related molecules: bis(dithiolene)molybdenum(IV) and -tungsten(IV,VI) complexes with variant terminal ligands.

The X-ray absorption spectra at the molybdenum and selenium K-edges and the tungsten L2,3-edges are acquired for a set of 14 Mo(IV) and W(IV,VI) bis(dithiolene) complexes related to the active sites of molybdo- and tungstoenzymes. The set includes square pyramidal [MoIVL(S2C2Me2)2]- (L = O2-, R3SiO-, RO-, RS-, RSe-) and [WIV(OR)(S2C2Me2)2]-, distorted trigonal prismatic [MoIV(CO)(SeR)(S2C2Me2)2]- and [WIV(CO)L(S2C2Me2)2]- (L = RS-, RSe-), and distorted octahedral [WVIO(OR)(S2C2Me2)2]-. The dithiolene simulates the pterin-dithiolene cofactor ligand, and L represents a protein ligand. Bond lengths are determined by EXAFS analysis using the GNXAS protocol. Normalized edge spectra, non-phase-shift-corrected Fourier transforms, and EXAFS data and fits are presented. Bond lengths determined by EXAFS and X-ray crystallography agree to < or = 0.02 A as do the M-Se distances determined by both metal and selenium EXAFS. The complexes [MoIV(QR)(S2C2Me2)2]- simulate protein ligation by the DMSO reductase family of enzymes, including DMSO reductase itself (Q = O), dissimilatory nitrate reductase (Q = S), and formate dehydrogenase (Q = Se). Edge shifts of these complexes correlate with the ligand electronegativities. Terminal ligand binding is clearly distinguished in the presence of four Mo-S(dithiolene) interactions. Similarly, five-coordinate [ML(S2C2Me2)2]- and six-coordinate [M(CO)L(S2C2Me2)2]- are distinguishable by edge and EXAFS spectra. This study expands a previous XAS investigation of bis(dithiolene)metal(IV,V,VI) complexes (Musgrave, K. B.; Donahue, J. P.; Lorber, C.; Holm, R. H.; Hedman, B.; Hodgson, K. O. J. Am. Chem. Soc. 1999, 121, 10297) by including a larger inventory of molecules with variant physiologically relevant terminal ligation. The previous and present XAS results should prove useful in characterizing and refining metric features and structures of enzyme sites.

Transient dimer in the refolding kinetics of cytochrome c characterized by small-angle X-ray scattering.

The equilibrium unfolding and the kinetic refolding of cytochrome c (Cyt c) in the presence of imidazole were studied with small-angle X-ray scattering (SAXS). The equilibrium unfolding experiments showed the radius of gyration, R(g), of native Cyt c to swell approximately 1 A with the addition of imidazole. The thermodynamic parameter m also reflects an expansion of the protein as its lower value demonstrates an increase in solvent-accessible surface area over that of native Cyt c in the absence of imidazole. Refolding was studied in the presence of imidazole as it prevents misligated intermediate states from forming during the refolding process, simplifying the kinetics, and making them easier to resolve. Time-resolved decreases in the forward scattering amplitude, I(0), demonstrated the transient formation of an aggregated intermediate. Final protein and denaturant concentrations were varied in the refolding kinetics, and the singular value decomposition (SVD) method was employed to characterize the associated state. This state was determined to be a dimer, with properties consistent with a molten globule.

Investigation of the anomalous spectroscopic features of the copper sites in chicken ceruloplasmin: comparison to human ceruloplasmin.

Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.

Characterization of transient intermediates in lysozyme folding with time-resolved small-angle X-ray scattering.

We have used synchrotron radiation, together with stopped-flow and continuous-flow mixing techniques to monitor refolding of lysozyme at pH 5.2. From data measured at times which range from 14 ms to two seconds, we can monitor changes in the size, the shape and the pair distribution function of the polypeptide chain during the folding process. Comparison of the results with the properties of native and GdmCl-unfolded lysozyme shows that a major chain collapse occurs in the dead-time of mixing. During this process about 50 % of the change in radius of gyration between the unfolded protein and the native state occurs and the polypeptide chain adopts a globular shape. Time-resolved fluorescence spectra of this collapsed state suggest that the hydrophobic side-chains are still highly solvent accessible. A subsequently formed intermediate with helical structure in the alpha-domain is nearly identical in size and shape with native lysozyme and has a solvent-inaccessible hydrophobic core. Despite its native-like properties, this intermediate is only slightly more stable (DeltaG0=-4 kJ/mol) than the collapsed state and still much less stable than native lysozyme (DeltaDeltaG0=36 kJ/mol) at 20 degrees C.

Protein denaturation: a small-angle X-ray scattering study of the ensemble of unfolded states of cytochrome c.

Solution X-ray scattering was used to study the equilibrium unfolding of cytochrome c as a function of guanidine hydrochloride concentration at neutral pH. The radius of gyration (Rg) shows a cooperative transition with increasing denaturant with a similar Cm to that observed with circular dichroism. However, the lack of an isoscattering point in the X-ray scattering patterns suggests the equilibrium unfolding is not simply a two-state process. Singular value decomposition (SVD) analysis was applied to the scattering patterns to determine the number of distinct scattering species. SVD analysis reveals the existence of three components, suggesting that at least three equilibrium states of the protein exist. A model was employed to determine the thermodynamic parameters and the scattering profiles of the three equilibrium states. These scattering profiles show that one state is native (N). The other two states (U1, U2) are unfolded, with U2 being fully unfolded and U1 having some residual structure. Using the thermodynamic parameters to calculate fractional populations, U1 is maximally populated at intermediate denaturant concentrations while U2 is maximally populated at high denaturant concentrations. It is likely that there is a multiplicity of denatured states with U1 and U2 representing an average of the denatured states populated at intermediate and high denaturant concentrations, respectively.

Vanadium K-edge x-ray absorption spectroscopy reveals species differences within the same ascidian genera. A comparison of whole blood from Ascidia nigra and Ascidia ceratodes.

Vanadium K-edge x-ray absorption spectroscopy (XAS) was used to examine whole blood preparations from the tunicates Ascidia nigra and Ascidia ceratodes. Each XAS spectrum exhibits a rising edge inflection near 5480 eV characteristic of vanadium(III) and an intensity maximum at 5484.0 eV. In A. ceratodes blood cells, intrinsic aquo-VSO4+ complex ion is indicated by an inflection feature at 5476 eV in the first derivative of the vanadium K-edge XAS spectrum, but this feature is notably absent from the first derivative of the vanadium K-edge spectrum of blood cells from A. nigra. A strong pre-edge feature at 5468.6 eV also uniquely distinguishes the vanadium K-edge XAS spectrum of A. nigra blood cells, implying that vanadyl ion represents approximately 25% of the endogenous vanadium. However, the energy position of the rising edge inflection of the vanadium K-edge XAS spectrum of A. nigra (5479.5 eV) is 1 eV lower than that of A. ceratodes (5480.5 eV), the reverse of any expected shift arising from the endogenous vanadyl ion. Thus, in contrast to A. ceratodes, a significant fraction of the blood cell vanadium(III) in A. nigra is apparently in a ligation environment substantially different from that provided by water. These novel species-related differences may have taxonomic significance.

Spectroscopic and magnetic studies of human ceruloplasmin: identification of a redox-inactive reduced Type 1 copper site.

Ceruloplasmin is unique among the multicopper oxidases in that in addition to the usual copper stoichiometry of one Type 1 copper site and a Type 2/Type 3 trinuclear copper cluster, it contains two other Type 1 sites. This assignment of copper sites, based on copper quantitation, sequence alignment, and crystallography, is difficult to reconcile with the observed spectroscopy. Furthermore, some chemical or spectroscopic differences in ceruloplasmin have been reported depending on the method of purification. We have studied the resting (as isolated by a fast, one-step procedure) and peroxide-oxidized forms of human ceruloplasmin. Using a combination of X-ray absorption spectroscopy, a chemical assay, magnetic susceptibility, electron paramagnetic resonance spectroscopy, and absorption spectroscopy, we have determined that peroxide-oxidized ceruloplasmin contains one permanently reduced Type 1 site. This site is shown to have a reduction potential of approximately 1.0 V. Thus, one of the additional Type 1 sites in ceruloplasmin cannot be catalytically relevant in the form of the enzyme studied. Furthermore, the resting form of the enzyme contains an additional reducing equivalent, which is distributed among the remaining five copper sites as expected from their relative potentials. This may indicate that the resting form of ceruloplasmin in plasma under aerobic conditions is a four-electron oxidized form, which is consistent with its function in the four-electron reduction of dioxygen to water.